Electrophilic tripeptide analogs containing the ((D-phenylalanyl)prolyl)-arginyl-sequence are well known as effective inhibitors of the trypsin-like serine protease thrombin. H-(D)Phe-Pro-ArgCH.sub.2 Cl was first reported by Kettner and Shaw (Thromb. Res. 14, 969 (1979)) to be a selective but irreversible inhibitor of human thrombin. A number of studies looking for alternatives to the electrophilic P.sub.1 argininechloromethylketones that would yield a reversible protease inhibitor have been reported. Bajuez et al. (Folia Haematol. 109, s. 16 (1982)) found the corresponding aldehyde, D-phenylalanyl-prolyl-arginal, to be a reversible thrombin inhibitor with a K.sub.i =75 nM for human thrombin. The nitrile analog, D-phenylalanyl-prolyl-NHCH((CH.sub.2).sub.3 NHC(.dbd.NH)NH.sub.2)--CN, was found to be substantially less potent with a K.sub.i =700 nM (Kaiser et al., Pharmazie 46, 128 (1991)). A retroamide inhibitor, with the D-phenylalanyl-prolyl-sequence and 2-(4-guanidinophenylalanyl)-N-acetyl-2,2-difluoroethylamine substituting for an electrophilic arginine derivative, is a good inhibitor with a K.sub.i of 70 nM for thrombin (Altenburger and Schirlin, Tetrahedron Lett. 32, 7255 (1991)). Cheng et al. claim that the substitution of racemic diphenyl 1-amino-4-methoxybutylphosphonate for an electrophilic arginine derivative gives very good inhibitors with a K.sub.i =4.8 nM (Tetrahedron Lett. 32, 7333 (1991)). Iwanowicz et al. (Bioorgan. Med. Chem. Lett. 2, 1607 (1992)) has studied the efficacy of (D-phenylalanine)prolyl-conjugated to --NHCH[(CH.sub.2).sub.4 NH.sub.2 ]CH(OH)CO.sub.2 Me) and --NHCH[(CH.sub.2).sub.4 NH.sub.2 ]C(.dbd.O)CO.sub.2 Me derivatives. The most effective inhibitor of human thrombin reported to date is the boropeptide acetyl-D-phenylalanyl-prolyl-boro arginine with a K.sub.i =0.041 nM (Kettner et al., J. Biol. Chem. 265, 18289 (1990)).
Walker et al. (Biochem. J. 230, 645 (1985)) published a comparative study of irreversible thrombin inhibitors based on the D-pherlylalarlyl-prolyl-argininyl sequence confirming the earlier report by Kettner and Shaw (1979). H-(D)Phe-Pro-ArgCH.sub.2 Cl was found to be the most effective inhibitor (K.sub.i =25 nM) while replacing the D-phenylalanine with 4-amino-D-phenylalanine or .omega.-benzoyl-D-lysine gave less active analogs. Compounds in development include -(prolyl)arginal derivatives with a variety of unusual P.sub.3 amino acids including D-N-methylphenylglycine, Boc-D-fluorophenylglycine as well as constrained cyclized derivatives of D-phenylglycine and D-phenylalanine (Shuman et. al., J. Med. Chem. 36, 314 (1993)). Balasubramanian et al. (J. Med. Chem. 36, 300 (1993)) has reported an extensive study of replacements for the P.sub.3 D-phenylalanine of D-phenylalanyl-prolyl-arginal and found the dihydrocinnamoyl group to be effective, although somewhat less potent.
Patent disclosures in this area have centered around suitably protected peptides composed of natural and unnatural amino acids. In U.S. Pat. No. 5,187,157 DuPont Merck has disclosed peptides comprised of C-terminal boronic acid derivatives of lysine, ornithine and arginine as reversible inhibitors of trypsin-like serine proteases, as well as a series of boropeptides active as elastase inhibitors in U.S. Pat. No. 4,499,082. In European Patent Application EP 471 651 A2 Sandoz disclosed borolysine and boroarginine peptide analogs containing at least one unnatural hydrophobic .alpha.-amino acid substituted with groups such as the trimethylsilyl- or naphthyl-. In U.S. Pat. No. 5,106,948 was disclosed a series of boropeptides that are effective as cytotoxic agents. In PCT Application WO 92/07869, Thrombosis Research Institute has disclosed tripeptide analogs containing a P.sub.2 proline and an unnatural disubstituted amino acid at P.sub.3. A variety of electrophilic and non-electrophilic .alpha.-amino acid analogs were claimed as suitable P.sub.1 substituents. Tripeptide antithrombotic agents limited to .alpha.-alkyl and .alpha.-aryl or heteroaryl substituted glycines at P.sub.3 conjugated to -(prolyl)arginal were been disclosed by Lilly (European Patent Application EP 479 489 A2). Marion Merrell Dow disclosed a series of activated electrophilic ketone analogs of peptidase substrates useful for inhibiting serine-, carboxylic acid- and metallo-proteolytic enzymes; compounds are peptides composed of suitably protected .alpha.-amino acids conjugated to an electrophilic ketone derivative of an .alpha.-amino acid (European Patent Applications EP 417 721 A2, EP 364 344 A2, EP 363 284 A2, EP 195 212 A2). Astra has disclosed a series of .alpha.-((trifluoroethyl)oxymethyl)-arginine tripeptides (European Patent Application EP 0 530 167 A). Georgia Tech Research Corporation disclosed peptidyl ketoamides, -ketoacids and -ketoesters as inhibitors of serine and cysteine proteases (WO 92/12140). Boehringer Ingelheim disclosed a series of trifluoromethyl- and .alpha.,.alpha.-difluoromethyl-.beta.-ketoesterpeptide derivatives as elastase inhibitors (EP 0 369 391 A2).
The present invention concerns dipeptides which contain an electrophilic derivative of an .alpha.-amino acid at P.sub.1 conjugated with an N,N-disubstituted .alpha.-amino acid at P.sub.2. The electrophilic functional groups used to derivatize the P.sub.1 amino acid analog are: boronic acids and their esters, .alpha.-mono- and .alpha.-perhaloketones, vicinal di- and tricarbonyl compounds, and .alpha.,.alpha.-dihalo-g-ketoesters. The N,N-disubstituted .alpha.-amino acids are derivatives of an amino acid other than proline where the .alpha.-amino group is alkylated and acylated or diacylated to give alicyclic or cyclic substituents. As a result these compounds are found to have the advantage of an improved toxicological profile as well as the selectivity and inhibition activity for thrombin required for a useful therapeutic agent.